Atrial natriuretic peptide (ANP) is the first described peptide in a family of hormones which regulate body fluid homeostasis (see Brenner et al., 1990). The description of the potent diuretic and natriuretic properties of atrial extracts by de Bold et al. (1981) was the first evidence that the heart could be an endocrine organ. The subsequent isolation and characterization of this activity by groups including Flynn et al. (1981) characterized ANP as the first secreted cardiac hormone. ANP is secreted by atrial myocytes in response to increased intravascular volume. Once it is in the circulation, its effects are primarily on the kidney, vascular tissue, and adrenal gland, in which its actions lead to the excretion of sodium and water by the kidneys and a decrease in intravascular volume and blood pressure (Atlas et al., 1987).
Matsuo and his coworkers isolated two other natriuretic peptides. Brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) were both isolated from porcine brain extracts on the basis of their potent relaxant effects on chick rectum (Sudeh et al., 1988; Sudeh et al., 1990). BNP is of myocardial cell origin, and like ANP circulates in human plasma (de Bold et al., 1981; Burnett et al., 1984). BNP is natriuretic, renin inhibiting, vasodilating, and lusitropic (Mukoyama et al., 1991; Yamamoto et al. 1996; Grantham et al., 1996). CNP is of endothelial cell origin and functions as a vasodilating and growth-inhibiting peptide (Suga et al., 1992; Stingo et al., 1992; Koller et al., 1991). ANP and BNP are increased in the plasma and heart during congestive heart failure (CHF) in humans, and they exert important cardiorenal protective actions in addition to serving as serum markers for ventricular dysfunction (Stevens et al., 1995; Yamamoto et al., 1997; McDonagh et al., 1998).
ANP, BNP and CNP are synthesized from large precursor proteins, and the mature, active peptides have a 17 amino acid loop formed by an intramolecular disulfide linkage. In the human peptides, eleven of these amino acids are identical in ANP, BNP, and CNP, whereas thexe2x80x94and C-terminal tails vary in both length and composition (see Kambayashi et al., 1990; and Tawaragi et al., 1991). CNP has no C-terminal tail, and studies of the structure of the gene for CNP demonstrated that translation is terminated by a stop codon immediately after the final cysteine codon in the mRNA.
Among species, the amino acid sequence of both ANP and CNP are highly conserved, whereas the structure of BNP varies greatly. For example, the mature 28 amino acid human and porcine ANPs are identical, and there is only one substitution in the rat peptide. The existence of this structural variation, coupled with the presence of at least three types of receptors specific for the natriuretic peptides, suggests that the physiological control of body fluid homeostasis is complex. ANP and CNP both decrease cardiac preload. However, unlike ANP, CNP is not natriuretic (Stingo et al., 1992).
The diverse actions of ANP, BNP and CNP on both the cardiovascular system and the kidney, as well as their roles in pathophysiological states such as heart failure, hypertension, and renal disease, have made the native peptides and their analog molecules of great interest to both clinical and basic scientists. See, for example, Lewicki et al. (U.S. Pat. Nos. 5,114,923, 4,804,650 and 4,757,048), Johnson et al. (U.S. Pat. No. 5,047,397) and Johnson et al. (U.S. Pat. No. 4,935,492), and Wei et al. (U.S. Pat. No. 5,583,108). U.S. Pat. No. 5,583,108 relates to a chimera of ANP and CNP, termed vasonatrin peptide (VNP). VNP, which includes 22 amino acids of CNP and the 5 amino acids at the carboxy-terminus of ANP, has arterial and venous vasodilating and natriuretic effects.
A fourth natriuretic peptide (NP), Dendroaspis natriuretic peptide (DNP), possesses structural similarity to ANP, BNP, and CNP. Isolated from the venom of Dendroaspis angusticeps or green mamba snake, DNP is a 38 amino acid peptide that contains a 17 amino acid disulfide ring structure similar to that of ANP, BNP, and CNP (FIG. 1), all of which mediate biologic actions through particulate guanylyl cyclase receptors and generation of cyclic guanosine monophosphate (cGMP) (Schweitz et al., 1992). DNP vasorelaxes rodent aorta and isolated canine coronary arteries with potency comparable to that of ANP (Schweitz et al., 1992; Wennberg et al., 1997). Additionally, DNP substantially augments the formation of cGMP, the second messenger for the other natriuretic peptides, in aortic endothelial cells (Schweitz et al., 1992).
Thus, there is a continuing need to identify peptides with properties such as those of natriuretic peptides which are useful to prevent or treat cardiovascular disorders, e.g., congestive heart failure.
The present invention provides an isolated and purified peptide compound having natriuretic, renin-suppressing, diuretic and/or vasodilator activity in mammals. Preferably, the peptide comprises a compound of formula (I):
X0-Pro-X1-A5-A1-A3-Pro-A1-Pro-A1-A5-Pro-X1-X1-X1-A4xe2x80x83xe2x80x83(SEQ ID NO:4)
wherein A1 is Leu, Lys, Arg, His, Orn, Asn or Gln; A3 is Asp or Glu; A4 is Lys, Arg, Orn, Ala, Thr, Asn, or Gln; A5 is Gly, Ala, Val, Met, Leu, Norleucine or Ile; X0 is absent or is a peptide of from 1 to 35 amino acid residues, preferably of from 1 to 25 amino acid residues, which peptide has a Cys residue at the C-terminus, and more preferably residues from the N-terminus of BNP or CNP; and X1 is Ser or Thr; or a compound of formula (II):
X0-Pro-X1-A5-A1-A3-Pro-A1-Pro-A1-A5-Pro-X1-X1-X1-A4-X2xe2x80x83xe2x80x83(SEQ ID NO:12)
wherein A1 is Leu, Lys, Arg, His, Orn, Asn or Gln; A3 is Asp or Glu; A4 is Lys, Arg, Orn, Ala, Thr, Asn, or Gln; A5 is Gly, Ala, Val, Met, Leu, Norleucine or Ile; X2 is absent or is a peptide of from 1 to 35 amino acid residues, preferably of from 1 to 25 amino acid residues; X0 is absent or is a peptide of from 1 to 35 amino acid residues, preferably of from 1 to 25 amino acid residues, which peptide has a Cys residue at the C-terminus, and more preferably residues residues from the N-terminus of BNP or CNP; and X1 is Ser or Thr.
One preferred peptide of the invention includes a chimeric peptide which is a 41 amino acid peptide combining the core ring structure of BNP with the C-terminus of DNP. Thus, a preferred compound of formula (I) is a chimeric peptide comprising Ser-Pro-Lys-Met-Val-Gln-Gly-Ser-Gly-Cys-Phe-Gly-Arg-Lys-Met-Asp-Arg-Ile-Ser-Ser-Ser-Ser-Gly-Leu-Gly-Cys-Pro-Ser-Leu-Arg-Asp-Pro-Arg-Pro-Asn-Ala-Pro-Ser-Thr-Ser-Ala (SEQ ID NO:1; BD-NP; see FIG. 4), or a biologically active variant or fragment thereof. Preferably, the chimeric peptide has a disulfide bridge between Cys 10 and Cys 26. Other preferred peptides of the invention include a 37 amino acid peptide combining the core ring structure of CNP with the C-terminus of DNP. Thus, another preferred compound of formula (I) is a chimeric peptide comprising Gly-Leu-Ser-Lys-Gly-Cys-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Met-Ser-Gly-Leu-Gly-Cys-Pro-Ser-Leu-Arg-Asp-Pro-Arg-Pro-Asn-Ala-Pro-Ser-Thr-Ser-Ala (SEQ ID NO:2; CD-NP; see FIG. 4), or a biologically active variant or fragment thereof. Preferably, the chimeric peptide has a disulfide bridge between Cys 6 and Cys 22. Thus, X0, if present, is preferably the N-terminus of human BNP, i.e., Ser-Pro-Lys-Met-Val-Gln-Glu-Ser-Gly-Cys-Phe-Gly-Arg-Lys-Met-Asp-Arg-Ile-Ser-Ser-Ser-Ser-Gly-Leu-Gly-Cys (SEQ ID NO:7), or the N-terminus of human CNP, i.e., Gly-Leu-Ser-Lys-Gly-Cys-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Met-Ser-Gly-Leu-Gly-Cys (SEQ ID NO:8). Yet another preferred peptide includes a portion of the carboxy-terminus of DNP, preferably which includes the carboxy-terminal 15 amino acids (SEQ ID NO:3; see FIG. 4), or a biologically active variant or fragment thereof. As used herein, the term xe2x80x9cbiologically activexe2x80x9d means that a peptide of the invention has at least one of the activities of a native natriuretic peptide. In one embodiment, the compound of formula (I) is a variant of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3, i.e., the compound is not SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.
Also provided is a compound which comprises a variant of a peptide fragment of Dendroaspis natriuretic peptide (SEQ ID NO:10), i.e., a variant of SEQ ID NO:3, wherein the variant has one or more amino acid substitutions selected from the group consisting of Gly to Pro; Glu to Asp; Val to Ile, Leu, Met, Phe, Ala or Nor; Met to Leu, Phe or Ile; Lys to Arg, Gln or Asn; Tyr to Trp, Phe, Thr or Ser; Asp to Glu; Phe to Leu, Val, Ile or Ala; His to Asn, Gln, Lys or Arg; Ile to Leu, Val, Met, Phe, Nor or Ala; Cys to Ser; Pro to Gly; Ser to Thr; Thr to Ser; Arg to Lys, Gln or Asn; Ala to Val, Ile, or Leu; Leu to Nor, Ile, Val, Met, Ala, or Phe; Asp to Glu; and Asn to Gln, His, Lys or Arg. In one embodiment, the compound comprises a variant of a peptide fragment of SEQ ID NO: 3, wherein the variant has one or more amino acid substitutions selected from the group consisting of Pro to Gly; Ser to Thr; Thr to Ser; Arg to Lys; Gln or Asn; Ala to Val, Ile, or Leu; Leu to Nor, Ile, Val, Met, Ala, or Phe; Asp to Glu; and Asn to Gln, His, Lys or Arg. More preferably, the variant has one or more amino acid substitutions selected from the group consisting of Ser to Thr; Thr to Ser; Arg to Lys; Gln or Asn; Ala to Val, Ile, or Leu; Leu to Nor, Ile, Val, Met, Ala, or Phe; Asp to Glu; and Asn to Gln, His, Lys or Arg.
As described below, BD-NP has a combined effect in vivo, which includes potent vasodilatation with a focus on pulmonary vasodilation, natriuresis and suppression of renin. For example, in normal mammals, the administration of BD-NP significantly increases glomerular filtration rate (GFR), decreases proximal fractional reabsorption of sodium (PFRNa), and more strongly suppresses plasma renin activity, relative to the administration of DNP. Further, in normal mammals, the administration of BD-NP (e.g., at 50 ng/kg/minute) has no effect on renal blood flow (RBF), increases urinary cGMP excretion (UcGMPV), has a potent renin suppressing effect, more potently decreases mean arterial pressure (MAP), and more potently decreases right atrial pressure (RAP) and pulmonary capillary pressure (PCWP) with more potent pulmonary vasodilatation, relative to the administration of BNP.
As also described herein below, DNP-like immunoreactivity (DNP-LI) was present in human plasma and in the atrial myocardium, as well as in human urine. Moreover, DNP-LI was increased in human plasma in patients with CHF. DNP is also present in other mammalian species, e.g., in the canine plasma, urine and myocardium. In vivo, DNP is a very powerful stimulator of plasma and urinary cGMP generation and has potent natriuretic, diuretic, vasodilatory and renin-suppressing properties (Lisy et al., 1999b). Further, DNP shows therapeutic efficacy in normal canine (see Lisy et al., 1999b) as well as in a canine model of experimental heart failure (Lisy et al., 1999a).
As further described hereinbelow, the exogenous administration of DNP to dogs with mild or overt congestive heart failure resulted in decreases in cardiac filling pressures and mean arterial pressure, preserves cardiac output and increases glomerular filtration rate. Thus, the present invention provide a method to treat congestive heart failure which comprises the administration of DNP or a biologically active portion thereof, a peptide which is a chimeric natriuretic peptide of DNP, or a biologically active variant or fragment thereof.
Thus, the present invention also provides a composition useful as a natriuretic, renin-suppressor, diuretic and/or vasodilator. The composition comprises a therapeutically effective amount of at least one peptide of the invention in combination with a pharmaceutically acceptable carrier. Therefore, the invention further provides a method for inducing natriuresis, diuresis or vasodilation in a mammal, e.g., a human. The method comprises administering to the mammal a pharmaceutically effective amount of compound or composition of the invention. The present peptides may be useful, either singly or in combination, to treat (ameliorate or prevent) a number of pathological conditions, including congestive heart failure, acute or chronic kidney failure, hypertension, cirrhosis of the liver, nephrotic syndrome, and other edematous states.